U.S. patent application number 11/589516 was filed with the patent office on 2007-07-19 for west nile vaccine.
This patent application is currently assigned to Wyeth. Invention is credited to Hsien-Jue Chu.
Application Number | 20070166325 11/589516 |
Document ID | / |
Family ID | 27613166 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166325 |
Kind Code |
A1 |
Chu; Hsien-Jue |
July 19, 2007 |
West Nile vaccine
Abstract
The present invention provides a safe and effective vaccine
composition against West Nile virus disease. An immunogenically
active component of West Nile virus or plasmid DNA, an adjuvant
such as a metabolizable oil, and a pharmacologically acceptable
carrier are formulated into an immunizing vaccine. The invention
also provides a method for the prevention or amelioration of West
Nile disease, such as encephalitis, in equidae by administering the
vaccine composition herein set forth.
Inventors: |
Chu; Hsien-Jue; (Overland
Park, KS) |
Correspondence
Address: |
WYETH;PATENT LAW GROUP
5 GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
Wyeth
Madison
NJ
|
Family ID: |
27613166 |
Appl. No.: |
11/589516 |
Filed: |
October 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10202716 |
Jul 25, 2002 |
7153513 |
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11589516 |
Oct 30, 2006 |
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60308334 |
Jul 27, 2001 |
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Current U.S.
Class: |
424/204.1 ;
424/130.1 |
Current CPC
Class: |
A61K 2039/55566
20130101; A61K 39/12 20130101; A61K 2039/70 20130101; A61P 31/12
20180101; A61K 39/08 20130101; C12N 2770/24134 20130101; A61P 25/00
20180101; A61P 31/00 20180101; A61K 2039/552 20130101; A61K 39/39
20130101; A61K 9/1075 20130101; A61K 2039/53 20130101; C12N
2710/16734 20130101; A61P 31/04 20180101; A61K 2039/5252 20130101;
C12N 2760/16134 20130101; A61K 2039/5254 20130101; C12N 2770/36134
20130101 |
Class at
Publication: |
424/204.1 ;
424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/12 20060101 A61K039/12 |
Claims
1. A safened vaccine composition which comprises: an effective
immunizing amount of an immunogenically active component selected
from the group consisting of an inactivated whole or subunit West
Nile virus, an antigen derived from said virus, DNA derived from
said virus, and a mixture thereof; an immunogenically stimulating
amount of a metabolizable oil; and a pharmacologically acceptable
carrier.
2. The composition according to claim 1 wherein the immunogenically
active component is an inactivated whole or subunit West Nile
virus, and further wherein a dosage unit of said vaccine comprises
about 1.times.10.sup.4 TCID.sub.50 to about 1.times.10.sup.8
TCID.sub.50 of said virus.
3. The composition according to claim 1 wherein the metabolizable
oil is SP oil.
4. The composition according to claim 2 wherein the immunogenically
active component is an inactivated whole West Nile virus.
5. The composition according to claim 3 wherein said oil is present
in the amount of about 4% to 10% vol/vol.
6. The composition according to claim 4 wherein said virus is
present in sufficient quantity to provide at least about
1.times.10.sup.4 TCID.sub.50 per unit dose of said composition.
7. The composition according to claim 5 wherein said oil is present
in the amount of about 5% vol/vol.
8. The composition according to claim 7 wherein said virus is
present in sufficient quantity to provide at least about
1.times.10.sup.6 TCID.sub.50 per unit dose.
9. The composition according to claim 8 wherein said virus is
inactivated whole West Nile virus.
10. A method for the prevention or amelioration of West Nile
encephalitis in equidae which comprises administering to said
equidae a safened vaccine composition which comprises an effective
immunizing amount of an immunogenically active component selected
from the group consisting of an inactivated whole or subunit West
Nile virus, an antigen derived from said virus, DNA derived from
said virus, and a mixture thereof; an immunogenically stimulating
amount of a metabolizable oil; and a pharmacologically acceptable
carrier.
11. The method according to claim 10 having said vaccine
composition wherein the immunogenically active component is an
inactivated whole or subunit West Nile virus.
12. The method according to claim 10 having said vaccine
composition wherein the metabolizable oil is SP oil.
13. The method according to claim 10 wherein said equidae are
horses.
14. The method according to claim 13 wherein said horses are
pregnant mares.
15. The method according to claim 10 wherein said vaccine
composition is administered parenterally.
16. The method according to claim 10 wherein said vaccine
composition is administered intramuscularly.
17. The method according to claim 12 having a vaccine composition
wherein the immunogenically active component is inactivated whole
West Nile virus.
18. The method according to claim 17 wherein said virus is present
in an amount sufficient to provide at least about 1.times.10.sup.4
TCID.sub.50 and up to about 1.times.10.sup.9 TCID.sub.50 per unit
dose.
19. The method according to claim 18 wherein said virus is present
in an amount sufficient to provide at least about 1.times.10.sup.6
TCID.sub.50 per unit dose, and at least two doses are administered
to each said equidae.
20. The method according to claim 19 wherein the metabolizable oil
is SP oil and is present in an amount of about 5% vol/vol.
21. The composition according to claim 6 further comprising another
vaccine component directed against rabies virus, Eastern equine
encephalitis virus, Western equine encephalitis virus, Venezuelan
equine encephalitis virus, equine herpes virus such as EHV-1 or
EHV-4, Ehrlichia risticii, Streptococcus equi, tetanus toxoid,
equine influenza virus (EIV).
22. A vaccine composition, comprising: a) at least about
1.times.10.sup.4 TCID.sub.50 and up to about 1.times.10.sup.9
TCID.sub.50 per unit dose of inactivated West Nile virus; and b)
about 4% to 10% vol/vol of a metabolizable oil adjuvant comprising
about 1 to 3% polyoxyethylene-polyoxypropylene block copolymer,
about 2 to 6% of squalane and about 0.1 to 0.5% of polyoxyethylene
sorbitan monooleate.
23. A safened and effective West Nile virus vaccine for equidae,
comprising: a) at least about 1.times.10.sup.6 TCID.sub.50 to about
1.times.10.sup.8 TCID.sub.50 per unit dose of killed or inactivated
West Nile virus, and b) at least about 1% vol/vol of an adjuvant
comprising at least one metabolizable oil and at least one wetting
or dispersing agent.
24. The vaccine of claim 23, wherein said vaccine comprises at
least about 1.times.10.sup.7 TCID.sub.50 of said virus.
25. The vaccine of claim 24, wherein said vaccine comprises at
least about 5.times.10.sup.7 TCID.sub.50 of said virus.
26. The vaccine of claim 23, wherein said vaccine is formulated
into two dosage units.
27. The vaccine of claim 23, comprising at least about 4% of said
adjuvant.
28. The vaccine of claim 27, comprising about 4% to 10% of said
adjuvant.
29. The vaccine of claim 23, wherein said adjuvant is SP oil.
30. The vaccine of claim 23, comprising at least two wetting or
dispersing agents.
31. The vaccine of claim 30, wherein said wetting or dispersing
agents are selected from the group consisting of non-ionic
surfactants.
32. The vaccine of claim 31, wherein said non-ionic surfactants are
selected from the group consisting of
polyoxyethylene/polyoxypropylene block copolymers and
polyoxyethylene esters.
33. A vaccine regimen for horses or other equidae, comprising two
dosage units of killed or inactivated West Nile virus, wherein each
said dosage unit comprises about 0.5 to 5 milliliters of a
composition containing at least about 5.times.10.sup.7 TCID.sub.50
of said virus and about 1 to 10% vol/vol of an adjuvant, said
adjuvant comprising at least one metabolizable oil and at least two
nonionic surfactants, and further wherein said dosage unit
comprises a pharmacologically acceptable carrier.
34. The vaccine regimen of claim 33, wherein said adjuvant is SP
oil.
35. The vaccine regimen of claim 34, wherein said SP oil comprises
squalane in an amount of about 2 to 6% by weight of said adjuvant,
about 1 to 3% of polyoxyethylene/polyoxypropylene block copolymer
by weight of said adjuvant, and about 0.1 to 0.5% of
polyoxyethylene sorbitan monooleate by weight of said adjuvant.
36. The vaccine regimen of claim 33, further comprising a vaccine
component directed to at least one member selected from the group
consisting of influenza virus, Eastern, Western and Venezuelan
rhinopneumonitis virus and tetanus toxoid.
37. A West Nile virus vaccine composition comprising West Nile
plasmid DNA in an amount of about 50 to 3,000 micrograms per
dose.
38. The composition of claim 37, wherein said composition comprises
about 100 to 1,000 micrograms per dose.
39. The composition of claim 38, wherein said composition comprises
about 100 to 250 micrograms per dose.
40. A West Nile virus vaccine regimen which comprises administering
to a horse at least about one dose of the composition of claim
37.
41. The regimen of claim 40, which comprises administering at least
about two doses.
Description
[0001] This application claims priority from co-pending U.S.
application Ser. No. 60/308,334 filed on Jul. 27, 2001.
FIELD OF THE INVENTION
[0002] The present invention relates to safe and effective West
Nile Virus vaccines, and to methods of administering same to
mammals, in particular horses.
BACKGROUND OF THE INVENTION
[0003] Known as a Flavivirus, the West Nile virus was first
identified in 1937 in Africa and first found in North America in
1999. Migratory birds are considered the primary means whereby
infection is spread within and between countries. The virus is
transmitted by mosquitoes that have acquired infection by feeding
on viremic birds. The virus is then amplified during periods of
adult mosquito blood-feeding. Infected mosquitos then transmit the
virus to humans and animals upon feeding thereon.
[0004] West Nile virus is the causative agent for West Nile Virus
disease, particularly West Nile encephalitis, predominately in
humans, other mammals and birds. The chief concern in both the
United States and foreign countries is the lack of effective
treatment for West Nile virus disease. Anti-inflammatory drugs are
used to combat swelling of central nervous system tissues, but
beyond that no medical intervention is available. Nor is there
believed to be a suitable vaccine known to prevent the infection.
To date, preventing contact with carriers appears to be the only
means of controlling the West Nile virus.
[0005] What is therefore needed in the art is to provide a safe and
effective equine West Nile virus vaccine composition. The vaccine
composition should be sufficiently safened so as to be suitable for
administration even to pregnant mares without adverse effect.
[0006] Also needed is a method for the prevention or amelioration
of West Nile Virus disease, particularly West Nile encephalitis, in
equidae and other mammals.
SUMMARY OF THE INVENTION
[0007] The present invention provides a safened vaccine composition
which comprises: an effective immunizing amount of an
immunogenically active component selected from the group consisting
of an inactivated whole or subunit West Nile virus, an antigen
derived from said virus, DNA derived from said virus, and a mixture
thereof; an immunogenically stimulating amount of a metabolizable
oil; and a pharmacologically acceptable carrier.
[0008] The present invention also provides a method for the
prevention or amelioration of West Nile encephalitis in equidae
which comprises administering to said equidae a safened vaccine
composition which comprises an effective immunizing amount of an
immunogenically active component selected from the group consisting
of an inactivated whole or subunit West Nile virus, an antigen
derived from said virus, DNA derived from said virus, and a mixture
thereof; an immunogenically stimulating amount of a metabolizable
oil; and a pharmacologically acceptable carrier.
[0009] Also provided as part of the invention is a safe and
effective West Nile Virus vaccine suitable for use in horses which
comprises an immunogenically active component selected from the
group consisting of an inactivated whole or subunit West Nile
virus, an antigen derived from said virus, DNA derived from said
virus, and a mixture thereof; an immunogenically stimulating amount
of a metabolizable oil; and a pharmacologically acceptable
carrier.
[0010] In a further embodiment, the invention describes a vaccine
composition comprising at least about at least about
1.times.10.sup.4 TCID.sub.50 per unit dose of inactivated West Nile
virus, or a component thereof, and about 4% to 10% vol/vol of a
metabolizable oil comprising about 1 to 3%
polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% of
squalane and about 0.1 to 0.5% of polyoxyethylene sorbitan
monooleate.
[0011] Further provided is a safened and effective West Nile virus
vaccine for equidae, comprising at least about 1.times.10.sup.6
TCID.sub.50 per unit dose of killed or inactivated West Nile virus,
and at least about 1% vol/vol of an adjuvant comprising at least
one metabolizable oil and at least one wetting or dispersing
agent.
[0012] The invention also sets forth a vaccine regimen for horses,
comprising two dosage units of killed or inactivated West Nile
virus, wherein each said dosage unit comprises about 0.5 to 5
milliliters of a composition containing at least about
5.times.10.sup.7 TCID.sub.50 of said virus and about 1 to 10%
vol/vol of an adjuvant, said adjuvant comprising at least one
metabolizable oil and at least two nonionic surfactants, and
further wherein said dosage unit comprises a pharmacologically
acceptable carrier.
[0013] Also provided as part of the invention is an equine vaccine
containing West Nile plasmid DNA in an amount of about 50 to 3,000
micrograms per dose, together with one or more adjuvants and a
suitable carrier. A vaccine regimen would comprise administering at
least about one dose of this composition, and desirably at least
about 2 doses, to horses for immunization against West Nile virus
disease.
[0014] Further, objects and features of the invention will become
apparent from the detailed description and the claims set forth
herein below.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Scientists believe that the West Nile virus follows the same
pattern of infection found with other mosquito-transmitted viruses.
When an infected mosquito bites an equid, the virus enters the skin
or tissues just below the bite site, where it is picked up by the
circulation. The virus can multiply in the bloodstream, and the
equid may develop a fever, which often goes undetected because
there are no other signs of illness at the time. However, once the
virus has invaded the nervous system, clinical signs appear within
one to three days. Most affected equidae, such as horses, first
exhibit signs of posterior weakness or paralysis and poor
coordination. Depression and related behavior changes may accompany
the physical changes. In severe cases, tremors, convulsions,
paddling of the limbs and paralysis may develop. Severe
neurological problems and mortality have also been observed. To
date, no vaccine known to prevent West Nile virus in equidae is
available; and the only means of controlling the West Nile virus
appears to be the prevention of contact with a carrier.
[0016] It has now been found that a safe and effective vaccine
composition which comprises an effective immunizing amount of an
immunogenically active component selected from the group consisting
of an inactivated whole or subunit West Nile virus, an antigen
derived from said virus, DNA derived from said virus, plasmid West
Nile virus DNA, plasmid with sequence inserts of said virus, and a
mixture thereof; an immunogenically stimulating amount of an
adjuvant, in particular a metabolizable oil; and a
pharmacologically acceptable carrier may be administered to
equidae, particularly horses, to prevent or ameliorate West Nile
Virus disease such as encephalitis.
[0017] DNA derived from the West Nile virus may be obtained via
isolation from sources such as the fluids or tissues of equine or
avian species diagnosed to have West Nile encephalitis. Such
sources include cerebral spinal fluid or sections of spinal cord or
brain. DNA may also be obtained using other available techniques
such as plasmid technology. For example, suitable cells of an
organism, e.g. E. coli, may be transformed with a plasmid
containing West Nile protein sequence inserts to obtain a master
seed. The master seed may then be cultured and passaged.
Transformed cells containing the West Nile DNA may then be
harvested, and the DNA isolated and obtained using techniques
available to the skilled artisan.
[0018] Whole or subunit West Nile virus may be isolated from
infected animals using conventional techniques. Samples of the
virus may also be obtained from tissue culture collections which
maintain a depository for organisms such as West Nile. At the
American Type Culture Collection (ATCC), for example, the West Nile
virus has been deposited under ATCC No.s VR-82, VR-1267 and VR-1267
AF.
[0019] Whole or subunit West Nile virus may be inactivated by
conventional inactivating means, for example chemical inactivation
using chemical inactivating agents such as binary ethyleneimine,
beta-propiolactone, formalin, gluteraldehyde, sodium dodecyl
sulfate, or the like or a mixture thereof, preferably formalin.
Said virus may also be inactivated by heat or psoralen in the
presence of ultraviolet light. (Live, attenuated West Nile virus
may also be used in certain embodiments, but this is perhaps much
less preferred.)
[0020] Whole or subunit West Nile virus may be maintained or grown
in mouse brains or in suitable tissue culture media, such as
optiMEM (LTI, Grand Island, N.Y.) or MEM media, or in cells known
in the art such as African green monkey kidney (Vero) cells or baby
hamster (BHK) cells, preferably Vero cells. Said virus may then be
separated from the tissue culture or cell media using conventional
techniques such as centrifugation, filtration or the like.
[0021] A preferred West Nile virus isolate may be obtained from the
National Veterinary Services Laboratory (part of the United States
Department of Agriculture) in Ames, Iowa as strain VM-2. The virus
strain may be plaque purified up to three times, and passaged to
X+5 in Vero cells.
[0022] As used herein the term "immunogenically active" designates
the ability to stimulate an immune response, i.e., to stimulate the
production of antibodies, particularly humoral antibodies, or to
stimulate a cell-mediated response. For example, the ability to
stimulate the production of circulating or secretory antibodies or
the production of a cell-mediated response in local mucosal
regions, (e.g., intestinal mucosa), peripheral blood, cerebral
spinal fluid or the like.
[0023] The effective immunizing amount of the immunogenically
active component may vary and may be any amount sufficient to evoke
an immune response and provide immunological protection against
West Nile Virus disease. Amounts wherein a dosage unit comprises at
least about 1.times.10.sup.4 TCID.sub.50 of killed or inactivated
whole or subunit virus cells or antigen or DNA cells derived
therefrom or a mixture thereof, preferably at least about
1.times.10.sup.6 TCID.sub.50, are suitable. Even more preferably,
at least about 1.times.10.sup.7 TCID.sub.50 per dosage unit may be
utilized. It is especially desirable that at least about
5.times.10.sup.7 TCID.sub.50 of killed or inactivated whole or
subunit West Nile virus cells or antigen or DNA cells derived
therefrom or a mixture thereof be used in the vaccine composition
of the invention. In certain embodiments, as much as
1.times.10.sup.9 TCID.sub.50 and more may be utilized. A range of
about 1.times.10.sup.4 TCID.sub.50 to about 1.times.10.sup.8
TCID.sub.50 may also be utilized.
[0024] In a further embodiment of the invention, it is contemplated
that about 50 to 3,000 micrograms (.mu.g or 10.sup.-6 grams) of
West Nile plasmid DNA may be utilized in one dosage unit of the
vaccine composition. More preferably, about 100 to 1,000 .mu.g may
be used, with about 100 to 250 .mu.g of plasmid DNA being more
preferred.
[0025] At least one dosage unit per animal is contemplated herein
as a vaccination regimen. In some embodiments, two or more dosage
units may be especially useful. A dosage unit may typically be
about 0.1 to 10 milliliters of vaccine composition, preferably
about 0.5 to 5 milliliters, and even more preferably about 1 to 2
milliliters, with each dosage unit containing the heretofore
described quantity of virus or virus component. The skilled artisan
will quickly recognize that a particular quantity of vaccine
composition per dosage unit, as well as the total number of dosage
units per vaccination regimen, may be optimized, so long as an
effective immunizing amount of the virus or a component thereof is
ultimately delivered to the animal.
[0026] The West Nile virus vaccine composition of the invention may
also contain one or more adjuvants. As used herein the term
"adjuvant" refers to any component which improves the body's
response to a vaccine. The adjuvant will typically comprise about
0.1 to 50% vol/vol of the vaccine formulation of the invention,
more preferably about 1 to 50% of the vaccine, and even more
desirably about 1 to 20% thereof. Amounts of about 4 to 10% may be
even more preferred.
[0027] Suitable adjuvants can include immunostimulating oils such
as certain metabolizable oils. Metabolizable oils suitable for use
in the composition of the invention include oil emulsions, e.g., SP
oil (hereinafter described), Emulsigen (MPV Laboratories, Ralston,
NZ), Montanide 264,266,26 (Seppic SA, Paris, France), as well as
peanut oil and other vegetable-based oils, squalane (shark liver
oil) or other metabolizable oil which can be shown to be suitable
as an adjuvant in veterinary vaccine practice.
[0028] In addition, the adjuvant may include one or more wetting or
dispersing agents in amounts of about 0.1 to 25%, more preferably
about 1 to 10%, and even more preferably about 1 to 3% by volume of
the adjuvant. Particularly preferred as wetting or dispersing
agents are non-ionic surfactants. Useful non-ionic surfactants
include polyoxyethylene/polyoxypropylene block copolymers,
especially those marketed under the trademark PLURONIC.RTM. and
available from BASF Corporation (Mt. Olive, N.J.). Other useful
nonionic surfactants include polyoxyethylene esters such as
polyoxyethylene sorbitan monooleate, available under the trademark
TWEEN 80.RTM.. It may be desirable to include more than one, e.g.
at least two, wetting or dispersing agents in the adjuvant as part
of the vaccine composition of the invention.
[0029] Other components of the adjuvant may include such
preservative compounds as formalin and thimerosal in amounts of up
to about 1% vol/vol of the adjuvant.
[0030] As an adjuvant, SP oil is preferred. As used in the
specification and claims, the term "SP oil" designates an oil
emulsion comprising a polyoxyethylene-polyoxypropylene block
copolymer, squalane, polyoxyethylene sorbitan monooleate and a
buffered salt solution. In general, the SP oil emulsion will
comprise about 1 to 3% vol/vol of block copolymer, about 2 to 6%
vol/vol of squalane, more particularly about 3 to 6% of squalane,
and about 0.1 to 0.5% vol/vol of polyoxyethylene sorbitan
monooleate, with the remainder being a buffered salt solution.
[0031] When utilized, immunogenically stimulating amounts of SP oil
as adjuvant in the vaccine composition of the invention may vary
according to the immunogenically active component, the degree of
potential infectious exposure, method of administration of the
vaccine composition, the age and size of the equid, or the like. In
general, amounts of about 1% to 50% vol/vol, preferably about 4% to
10% vol/vol, and more preferably about 4% to 5% vol/vol of SP oil
are suitable.
[0032] In general, it is believed that a live virus vaccine may
potentially lack sufficient safety in a given target host, and that
a killed or inactivated virus vaccine may potentially lack the
ability to stimulate a sufficiently effective immunologic response.
Commonly, an adjuvant or immunogenically stimulating compound is
used in combination with a killed or inactivated virus in a vaccine
composition to obtain acceptable efficacy. However, safety to the
target host is often compromised by the addition of an adjuvant.
For example, pregnant animals many times have been known to have a
significantly higher rate of miscarriage after being administered a
killed or inactivated virus vaccine that contains an adjuvant.
[0033] It has now been found that when a suitable adjuvant, e.g. a
metabolizable oil preferably such as SP oil, is used in combination
with an immunogenically active component as described hereinabove,
the resultant West Nile vaccine composition is safened for use in
equidae, particularly horses, even for use in pregnant mares, while
demonstrating important efficacy as well. Thus, the invention
achieves the concomitant goals of effective immunization and
safety, especially for pregnant animals. This combination of active
immunogen and adjuvant is unheralded in the art.
[0034] Pharmacologically acceptable carriers suitable for use in
the vaccine composition of the invention may be any conventional
liquid carrier suitable for veterinary pharmaceutical compositions,
preferably a balanced salt solution or other water-based solution
suitable for use in tissue culture media. Other available carriers
may also be utilized.
[0035] Additional excipients available in the art may also be
included in the vaccine composition according to the various
embodiments heretofore described. For example, pH modifiers may be
utilized.
[0036] The components of the vaccine composition of the invention
as heretofore described, including the carrier, may be combined
together using available techniques.
[0037] In addition to the immunogenically active component of West
Nile virus as described hereinabove as active ingredient, it is
contemplated that the vaccine composition of the invention may also
contain other active components such as an antipathogenic component
directed against rabies virus, Eastern equine encephalitis virus,
Western equine encephalitis virus, Venezuelan equine encephalitis
virus, equine herpes virus such as EHV-1 or EHV4, Ehrlichia
risticii, Streptococcus equi, tetanus toxoid, equine influenza
virus (EIV), or the like or a combination thereof. The quantities
of one or more of these viruses may be determined from efficacy
literature in the art, or determined using available
techniques.
[0038] In one embodiment of the invention the immunogenically
active component of the invention may be incorporated into
liposomes using known technology such as that described in Nature,
1974, 252, 252-254 or Journal of Immunology, 1978, 120, 1109-13. In
another embodiment of the invention, the immunogenically active
component of the invention may be conjugated to suitable biological
compounds such as polysaccharides, peptides, proteins, or the like,
or a combination thereof.
[0039] In a preferred embodiment of the invention, the inventive
vaccine composition may be formulated in dosage unit form as
heretofore described to facilitate administration and ensure
uniformity of dosage. Formulation may be effected using available
techniques, such as those applicable to preparations of
emulsions.
[0040] The inventive vaccine composition may be administered
parenterally, for example, intramuscularly, subcutaneously,
intraperitoneally, intradermally or the like, preferably
intramuscularly; or said composition may be administered orally or
intranasally.
[0041] Accordingly, the present invention also provides a method
for the prevention or amelioration of West Nile encephalitis in
equidae, preferably horses, which comprises administering to said
equidae a safened vaccine composition as described hereinabove.
[0042] In actual practice, the vaccine composition of the invention
is administered parenterally, subcutaneously, orally, intranasally,
or by other available means, preferably parenterally, more
preferably intramuscularly, in effective amounts according to a
schedule which may be determined by the time of anticipated
potential exposure to a carrier of the West Nile virus. In this
way, the treated animal may have time to build immunity prior to
the natural exposure. By way of non-limiting example, a typical
treatment schedule or dosing regimen may include parenteral
administration, preferably intramuscular injection of one dosage
unit, at least about 2-8 weeks prior to potential exposure. At
least two administrations are preferred, for example one dosage
unit at about 8 weeks and a second dosage unit at about 3-5 weeks
prior to potential exposure of the treated animal. As heretofore
set forth, a dosage unit will typically be within the range of
about 0.1 to 10 milliliters of vaccine composition containing the
previously described amounts of active and percentages of adjuvant
and inactive(s) as set forth. A dosage unit within the range of
about 0.5 to 5 milliliters is perhaps more preferred, with about 1
to 2 milliliter(s) being particularly preferred.
[0043] For a clearer understanding of the invention, the following
examples are set forth below. These examples are merely
illustrative and are not understood to limit the scope or
underlying principles of the invention in any way. Indeed, various
modifications of the invention, in addition to those shown and
described herein, will become apparent to those skilled in the art
from the following examples and the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
EXAMPLE 1
[0044] TABLE-US-00001 Preparation of Vaccine A/Formulation of SP
oil Ingredient Description Volume Polyoxyethylene-polyoxypropylene
block copolymer 20.0 ml (Pluronic .RTM. L121, BASF, Mt. Olive, NJ)
Squalane (Kodak, Rochester, NY) 40.0 ml Polyoxyethylenesorbitan
monooleate 3.2 ml (Tween .RTM. 80, Sigma Chemical, St. Louis, MO)
Buffered salt solution 936.8 ml (D-V PBS Solution, Ca, Mg free)
[0045] The ingredients are mixed and homogenized until a stable
mass or emulsion is formed. Prior to homogenization, the
ingredients or mixture may be autoclaved. The emulsion may be
further sterilized by filtration. Formalin may be added up to a
final concentration of 0.2%. Thimerosal may be added to a final
dilution of 1:10,000.
B/Vaccine Preparation
[0046] An equine isolate of West Nile virus, obtained from USDA
facilities in Ames, Iowa (Lot No. VM-2, Equine Origin, 1999 North
American isolate, second passage in VeroM cell culture), was
cultivated in multiple cultures of Vero cells in OptiMEM (LTI,
Grand Island, N.Y.) tissue culture medium at 37.degree. C. The
harvests are titrated and then inactivated by means of addition of
a 10% formalin solution to a final concentration of 0.1%. This is
allowed to inactivate at 37.degree. C. for a period of no less than
144 hours. Then, another addition of 0.1% formalin is added and
incubated at 37.degree. C. for another period of no less than 144
hours.
[0047] The vaccines are formulated by suspending the appropriate
volume of inactivated virus fluid in 1-20% by volume of SP oil per
1 mL dose.
EXAMPLE 2
Evaluation of Antibody Response to the Intramuscular Injection of
Test Vaccine
[0048] In this evaluation, horses are randomly divided into four
groups: one group of twenty horses is administered test vaccine at
a dose of 1.times.10.sup.7 TCID.sub.50 (Tissue Culture Infectious
Dose); a second group of twenty horses is administered test vaccine
at a dose of 5.times.10.sup.7 TCID.sub.50; a third group of five
horses is administered test vaccine at a dose of 1.times.10.sup.8
TCID.sub.50; and a fourth group of eight horses are maintained as
non-vaccinated environmental controls. Treated horses are given a
first dose of vaccine according to the group to which they are
assigned. At twenty-one days following administration of the first
dose, a second dose of the same vaccine is administered. All horses
are bled for serum at the time of administration of the first and
second dose and at weekly intervals through 28 days post second
dose administration. TABLE-US-00002 Test Vaccine A Component
Conc./Dose Volume/mL West Nile virus-Inactivated 1 .times. 10.sup.7
TCID.sub.50 0.0347 mL MEM.sup.1 NA 0.9138 mL SP oil 5% 0.0500 mL
Polymyxin B.sup.2 30.0 mcg/mL 0.0003 mL Neomycin 30.0 mcg/mL 0.0003
mL Thimerosal (5%) 1:20,000 0.0010 mL
[0049] TABLE-US-00003 Test Vaccine B Component Conc./Dose Volume/mL
West Nile virus-Inactivated 5 .times. 10.sup.7 TCID.sub.50 0.1734
mL MEM NA 0.7752 mL SP oil 5% 0.0500 mL Polymyxin B.sup.2 30.0
mcg/mL 0.0002 mL Neomycin.sup.3 30.0 mcg/mL 0.0002 mL
Thimerosal.sup.4 (5%) 1:20,000 0.0010 mL
[0050] TABLE-US-00004 Test Vaccine C Component Conc./Dose Volume/mL
West Nile virus-Inactivated 1 .times. 10.sup.8 TCID.sub.50 0.3467
mL MEM.sup.1 NA 0.6019 mL SP oil 5% 0.0500 mL Polymyxin B.sup.2
30.0 mcg/mL 0.0002 mL Neomycin.sup.3 30.0 mcg/mL 0.0002 mL
Thimerosal.sup.4 (5%) 1:20,000 0.0010 mL .sup.1LTI, Grand Island,
NY .sup.2Sigma, St. Louis, MO .sup.3Sigma, St. Louis, MO
.sup.4Sigma, St. Louis, MO
[0051] The serological data obtained is shown in Table I below,
wherein: 0 DPV 1 designates day zero, pre vaccination; and 14 DPV 2
designates day 14, post vaccination. NR designates no results.
[0052] As can be seen from the data on Table I, treated horses from
all groups showed significant increases in antibodies to West Nile
virus while the control horses maintained a non-detectable antibody
level. The level of response in the horses that received vaccine
was independent of the level of antigen in the vaccine that they
received. TABLE-US-00005 TABLE I Test Dose Test #1 Test #2 Vaccine
(TC1D.sub.50) 0DPV1 14DPV2 0DPV1 14DPV2 A 1 .times. 10.sup.7 <10
160 <10 80 A 1 .times. 10.sup.7 <10 20 <10 10 A 1 .times.
10.sup.7 <10 .gtoreq.320 <10 40 A 1 .times. 10.sup.7 <10
80 <10 .gtoreq.320 A 1 .times. 10.sup.7 <10 80 <10 40 A 1
.times. 10.sup.7 <10 80 <10 160 A 1 .times. 10.sup.7 <10
40 <10 40 A 1 .times. 10.sup.7 <10 80 <10 80 A 1 .times.
10.sup.7 <10 160 <10 80 A 1 .times. 10.sup.7 <10 40 <10
10 A 1 .times. 10.sup.7 <10 40 <10 10 A 1 .times. 10.sup.7
<10 160 <10 40 A 1 .times. 10.sup.7 <10 .gtoreq.320 <10
NR* A 1 .times. 10.sup.7 <10 .gtoreq.320 <10 NR A 1 .times.
10.sup.7 <10 .gtoreq.320 <10 80 A 1 .times. 10.sup.7 <10
160 <10 20 A 1 .times. 10.sup.7 .gtoreq.20 .gtoreq.320 <10
160 A 1 .times. 10.sup.7 <10 80 <10 80 A 1 .times. 10.sup.7
<10 80 <10 160 A 1 .times. 10.sup.7 <10 160 <10 160 B 5
.times. 10.sup.7 <10 80 <10 40 B 5 .times. 10.sup.7 <10 20
<10 <10 B 5 .times. 10.sup.7 <10 .gtoreq.320 <10 160 B
5 .times. 10.sup.7 <10 80 <10 .gtoreq.320 B 5 .times.
10.sup.7 <10 .gtoreq.320 <10 160 B 5 .times. 10.sup.7 <10
80 <10 80 B 5 .times. 10.sup.7 <10 160 <10 .gtoreq.320 B 5
.times. 10.sup.7 <10 80 <10 40 B 5 .times. 10.sup.7 <10
160 <10 40 B 5 .times. 10.sup.7 <10 40 <10 80 B 5 .times.
10.sup.7 <10 20 <10 20 B 5 .times. 10.sup.7 <10
.gtoreq.320 <10 160 B 5 .times. 10.sup.7 <10 .gtoreq.40
<10 .gtoreq.320 B 5 .times. 10.sup.7 <10 80 <10 40 B 5
.times. 10.sup.7 <10 80 <10 NR B 5 .times. 10.sup.7 <20
.gtoreq.320 <10 80 B 5 .times. 10.sup.7 .gtoreq.20 160 <10 80
B 5 .times. 10.sup.7 <10 .gtoreq.320 <10 160 B 5 .times.
10.sup.7 <10 .gtoreq.320 <10 .gtoreq.320 B 5 .times. 10.sup.7
<10 80 <10 160 C 1 .times. 10.sup.8 <10 .gtoreq.320 <10
.gtoreq.320 C 1 .times. 10.sup.8 <10 .gtoreq.320 <10 160 C 1
.times. 10.sup.8 <10 160 <10 80 C 1 .times. 10.sup.8 <10
.gtoreq.320 <10 NR C 1 .times. 10.sup.8 <10 40 <10 40
Control 0 <10 <10 <10 <10 Control 0 <10 <10
<10 <10 Control 0 <10 <10 <10 <10 Control 0
<10 <10 <10 NR Control 0 <10 <10 <10 <10
Control 0 <10 <10 <10 <10 Control 0 <10 <10
<10 <10 Control 0 <10 <10 <10 <10
EXAMPLE 3
Evaluation of Safety of Test Vaccine in Horses Under Field
Conditions
[0053] In this evaluation, 648 healthy male and female horses,
including 32 pregnant mares, are vaccinated with a 6.times.10.sup.6
TCID.sub.50 dose of inactivated test vaccine administered as a 1 mL
dose vaccination by intramuscular administration and followed in
three to four weeks by a second 1 mL dose vaccination. The treated
horses are housed and fed using conventional husbandry practices
for farm or stable. All treated horses are observed by a
veterinarian for 30 minutes following vaccination for immediate
reactions such as salivation, labored or irregular breathing,
shaking, or anaphylaxis. For two weeks post-vaccination, the horses
are observed daily for any delayed reactions such as lethargy,
anorexia or unusual swelling at the injection site. Blood samples
of 5 to 10 mL are taken by venipuncture from treated horses on the
day of first vaccination (day Zero) and at least once more at two
or more weeks post second vaccination (day 36 or greater).
Serological assays using PRNT.sup.5 testing are performed.
.sup.5Chang, G. J.; Hunt, A. R. and Davis B., Journal of Virology,
74 pp 4244-5422 (2000). TABLE-US-00006 Test Vaccine Component
Conc./Dose Volume/mL West Nile virus-Inactivated 6 .times. 10.sup.8
TCID.sub.50 0.21 mL SP oil 5% 0.05 mL MEM N/A 0.74 mL
[0054] No vaccine-induced safety problems were found in any of the
vaccinates, including the pregnant mares. This evaluation
demonstrates that the vaccine of the invention is safe for use in
horses under field conditions.
EXAMPLE 4
Evaluation of Efficacy of Test Vaccine (Multivalent and Monovalent
Preparations) in Horses Under Experimental Conditions
[0055] The efficacy of a combination vaccine containing killed West
Nile virus (WNV) against experimental WNV challenge was
evaluated.
[0056] A total of 30 horses were allotted into one vaccinated group
(20 horses) and one control group (10 horses). Horses in the
vaccinated group received intramuscularly two doses of the test
vaccine containing killed West Nile virus (5.times.10.sup.7
TCID.sub.50 per dose with 5% SP oil), influenza virus,
encephalomyelitis virus (Eastern, Western and Venezuelan),
rhinopneumonitis virus (serotypes 1 and 4), and tetanus toxoid,
three weeks apart. Serum samples were collected periodically for
antibody response measured by plaque reduction neutralization test
(PRNT). Twenty-four (24) days after the second vaccination, all
horses were challenged subcutaneously with WNV. After challenge,
horses were monitored for rectal temperature and any clinical signs
twice daily for two weeks and once weekly thereafter for detection
of viremia. Horses were euthanized and necropsied on 21 and 22 DPC.
Cerebrospinal fluid (CSF), spinal cord (cervical, thoracic, and
lumbar) and brain (frontal, occipital, medulla oblongata, and brain
stem) tissue samples were examined for gross pathology and
collected for virus isolation.
[0057] Fourteen days after the second vaccination, 75% of
vaccinated animals seroconverted (titer.gtoreq.5) with a geometric
mean titer of 10 while control animals remained negative
(titer<5). The vaccination conferred a significant protection
against viremia (a precursor to development of full-blown West Nile
Virus disease). Nine out of 10 (90%) controls developed viremia
after challenge, while only eight out of 20 (40%) vaccinates had
transient viremia, or viremia lasting only a few days at most.
Importantly, no WNV disease clinical signs were observed in any of
the challenged vaccinated animals throughout the observation
period. (Transient febrile responses were observed in one control
and two vaccinated horses. However, there was no evidence to
suggest these responses were due to WNV infection.) Petechial
hemorrhage in white matter and subdural hemorrhage were found in
the brain tissue from one control animal. WNV was isolated from the
brain but not from CSF and spinal cord samples collected from this
animal. No WNV was isolated from any of the tissue samples
collected from other challenged horses.
[0058] Results from this study demonstrated a significant
protection against both viremia and signs of clinical WNV disease
in horses vaccinated with the test combination vaccine.
[0059] A second study has conducted with a protocol similar to that
above, except that a monovalent WNV vaccine (WNV vaccine alone) was
utilized, and all horses were challenged with WNV at 12 months
after the second vaccination. Nine out of 11 (81.8%) of the
controls developed viremia after challenge, while only one out of
19 (5.3%) of the vaccinates had transient viremia. No WNV
associated clinical signs were observed in any of the challenged
animals throughout the observation period. No febrile responses
were observed in any of the challenged horses. No WNV was isolated
from any of the tissue or CSF samples collected from any of the
challenged horses. (Prior to challenge at the end of the 12 month
period, 17 of the nineteen vaccinated horses had plaque reduction
neutralization test (PRNT) titers of 5 or greater, while the
control group remained negative (<5).)
[0060] Results from this second study demonstrate a significant
protection (94% of preventable fraction) against viremia in horses
vaccinated with the killed monovalent WNV vaccine. These results
also demonstrate a long duration of protective immunity.
EXAMPLE 5
Evaluation of Efficacy of DNA Test Vaccine in Horses Under
Experimental Conditions
[0061] This example demonstrates the efficacy of a West Nile Virus
(WNV) DNA vaccine, as part of a further embodiment of the
invention. The DNA vaccine contained 100 .mu.g of purified DNA
adjuvanted with 5% SP oil per 2 mL dose, and was evaluated against
experimental WNV challenge.
[0062] For the composition of the WNV DNA vaccine, bacterial cells
were harvested from a culture passaged 10 times from a master seed
using E. coli DH10B obtained from Invitrogen (Carlsbad, Calif.)
containing a West Nile plasmid pCBWN obtained from the Centers for
Disease Control (Fort Collins, Colo.). The bacterial cells were
suspended in glucose-tris-EDTA buffer and lysed with sodium
hydroxide and sodium dodecyl sulfate. The lysate was neutralized
with a potassium acetate solution. The precipitated complex
material containing DNA, RNA, cell debris and proteins was removed
by filtration. The filtrate was precipitated with the addition of
isopropyl alcohol. The precipitate was collected by centrifugation
and resuspended in buffer. This process was repeated using ammonium
acetate. The precipitate collected was resuspended in buffer and
loaded into a chromatography column packed with Polyflo.RTM. resin.
The column was then washed and the plasmid DNA was eluted from the
column. The eluate was finally diafiltered extensively against
phosphate buffer saline. The purified plasmid DNA stocks were then
shipped for blending. The test vaccine contained 100 .mu.g of
plasmid DNA adjuvanted with 5% SP oil.
[0063] The horses used for testing were randomly assigned into two
groups: 20 animals received the WNV DNA vaccine, and 10 animals
were used as controls. The first group were vaccinated
intramuscularly with two 2.0 mL doses of vaccine three weeks apart.
The control horses received no vaccinations or placebos. One group
of horses (9 vaccinates and 5 controls) were challenged 5 weeks
after the second vaccination, whereas a second group of horses (11
vaccinates and 5 controls) were challenged 12 weeks after the
second vaccination. Briefly, Aedes albopictus mosquitoes which had
been infected with WNV 12 days prior to the horse challenge, were
allowed to feed on each horse for at least 5 minutes. Following
challenge, only those mosquitoes which were found to have engorged
with blood meals from each horse were frozen at -20.degree. C., and
the virus load was titrated as a pool subsequently. Mosquitoes were
then homogenized by vortexing using diluent. The homogenate was
centrifuged and the supernatant was removed for titration on Vero
cells.
[0064] After challenge, rectal temperatures were taken twice daily
for at least 9 days and clinical signs were monitored twice a day
for at least 21 days. Serum samples were taken twice a day for the
first 9 days post challenge (DPC); once daily from 10 to 14 DPC and
finally at 21 DPC. Virus isolation was performed on serum samples
from 0 DPC to 10 DPC for the first challenge group and from 0 DPC
to 11 DPC for the second challenge group. The first group of 14
horses and the second group of 16 horses were euthanized and
necropsied 28 to 37 DPC and 29 to 38 DPC, respectively.
Cerebrospinal fluid (CSF) and tissue samples from the cerebrum,
cerebellum and the brain stem were collected for gross pathology
and virus isolation.
[0065] At 14 days post second vaccination, 6 of 20 vaccinated had a
measurable titer of 2 or greater, and one horse had a titer of 5.
Vaccination had conferred protection against experimental West Nile
challenge using mosquitoes. Viremia was detected in 5 of 5 control
animals and 4 of 9 vaccinates in the first challenge group, whereas
4 of 5 controls and 2 of 11 vaccinates were viremic in the second
challenge group of horses. The viremia detected was transient, and
occurred only within the first six days after challenge. As a
whole, viremia was detected in 9 out of 10 (90%) control horses,
while only 6 out of 20 (30%) vaccinates were detected with
viremia.
[0066] No neurological signs attributable to WNV infection were
observed in any of the study horses throughout the challenge
observation period. No WNV was isolated from any of the tissue
samples collected from any of the study horses. One horse from the
first challenge group which was euthanized on 7 DPC showed no gross
or microscopic evidence of encephalitis or meningitis.
[0067] Results from this study demonstrated a significant
protection against viremia in horses vaccinated with the test
vaccine.
* * * * *